1. Field of the Invention
The present invention relates generally to the field of vascular leak, and particularly concerns toxins which induce or cause vascular leak syndrome (VLS). The invention provides immunotoxins (ITs) and cytokines which have been mutated to lack amino acid sequences which induce VLS and other toxic side effects. Disclosed are methods for mutating DNA segments encoding cytokines or immunotoxins so that an immunotoxin is produced that lacks sequences that induce VLS and other toxic side effects. Also disclosed are methods of preparing and using peptides which promote VLS and thus aid delivery of molecules into tissues. The present invention also relates to methods of preparing peptides which inhibit VLS and the used of mutated toxins as vaccines to protect immunized individuals from later toxicity.
2. Description of Related Art
VLS is often observed during bacterial sepsis and may involve IL-2 and a variety of other cytokines (Baluna and Vitetta, 1996). The mechanisms underlying VLS are unclear and are likely to involve a cascade of events which are initiated in endothelial cells (ECs) and involve inflammatory cascades and cytokines (Engert et al., 1997). VLS has a complex etiology involving damage to vascular endothelial cells (ECs) and extravasation of fluids and proteins resulting in interstitial edema, weight gain and, in its most severe form, kidney damage, aphasia, and pulmonary edema (Sausville and Vitetta, 1997; Baluna and Vitetta, 1996; Engert et al., 1997). Vascular leak syndrome (VLS) has been a major problem with all ITs thus far tested in humans, as well as cytokines such as interleukin 2 (IL-2), TNF and adenovirus vectors (Rosenberg et al., 1987; Rosensten et al., 1986).
ITs are hybrid molecules consisting of monoclonal antibodies (MAbs) or other cell-binding ligands, which are biochemically or genetically linked to toxins, toxin subunits, or ribosome inactivating proteins (RIPs) from plants, fungi or bacteria (Vitetta et al., 1993). Over the past two decades, ITs containing deglycosylated (dg) ricin A chain (dgRTA) have been developed, structurally optimized for stability and activity and evaluated for activity both in vitro, and in vivo in rodents, monkeys and humans (Vitetta et al., 1993; Sausville and Vitetta, 1997; Baluna and Vitetta, 1996).
It has been postulated that dgRTA-ITs induces VLS by damaging vascular endothelial cells (Soler-Rodriguez et al., 1993; Baluna et al., 1996). IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA) and other toxins, damage human ECs in vitro and in vivo (Dutcher et al., 1991; Rosenberg et al., 1987; Vial and Descotes, 1992). Studies using human umbilical vein ECs (HUVECs) demonstrated that dgRTA or ITs prepared with dgRTA can damage these cells within one hour (Soler-Rodriguez et al., 1993) while the inhibition of protein synthesis required 4 hrs or longer. DgRTA-ITs also interfere with fibronectin (Fn)-mediated adhesion (Baluna et al., 1996). Fn inhibits dgRTA-mediated damage to human umbilical vein endothelial cells (HUVECS) (Baluna et al., 1996). Cell adhesion to Fn is mediated by integrins which recognize RGD and LDV sequences in the Fn molecule (Makarem and Humphries, 1991; Wayner and Kovach, 1992).
Three MAbs linked to dgRTA have been evaluated in Phase I trials in over 200 patients with relapsed chemorefractory lymphoma, myeloma, Hodgins disease and graft vs. host disease (GVHD) (Sausville and Vitetta, 1997). These ITs have shown no evidence of myelotoxicity or hepatotoxicity, but all have induced VLS at the maximum tolerated dose (MTD) as defined by hypoalbuminemia, weight gain, and in the most severe cases, pulmonary edema and hypotension (Baluna et al., 1996). In addition, they have induced myalgia and, in 3% of patients, rhabdomyalyosis at the MTD (Sausville and Vitetta, 1997); this side effect may also be related to VLS and result from muscle edema. Further, aphesias have occurred in  less than 5% of patients"" these may be due to edema in the cerebral microvasculture.
In certain aspects, the invention provides the use of a modified proteinaceous composition that has altered, relative to the sequence of a native proteinaceous composition, at least one amino acid of a sequence comprising (x)D(y), for the manufacture of a medicament for the treatment of a disease, including but not limited to GVHD, non-Hodgkin""s and Hodgkin""s lumphoma, myeloma, as well as metastatic lesions of solid tumors and damage to endothelial cells (i.e., VLS).
Clearly, further development of dgRTA-ITs as well as other ITs containing toxins and RIPS, as well as cytokines as clinical agents would be greatly facilitated by the elimination or reduction of VLS. If VLS could be avoided or reduced it would permit the use of much higher doses of a variety of therapeutic agents such as Its, gene therapy and cytokines without the dose limiting side effects currently encountered.
The present invention overcomes the deficiencies in the art by providing methods for modulating the ability of various proteinacious compounds to induce toxic effects, and proteinacious compounds that have been modified such that they have modulated ability to induce toxic effects. In some embodiments, the invention allows for the production of ITs with a reduced ability to promote or induce such toxic effects, including, for example, VLS. ITs made in accordance with the invention are for any number of therapeutic applications, for example, the treatment of GVHD, non-Hodgkin""s and Hodgkin""s lymphoma, myloma, and metastatic lesions tumors, in some particular aspects solid tumors. The present invention also provides methods for reducing the VLS promoting ability of proteinaceous compositions through a mutation of sequences that induce or promote any of a number of toxic effects. The present invention provides ITs, IL-2 TNF and adenovirus with a reduced ability to promote toxic effects, and methods of using such compounds.
The invention, in one aspect, provides a method of modifying the ability of a proteinaceous composition to induce a toxic effect, comprising the steps of: identifying at least one amino acid sequence comprising the sequence (x)D(y), wherein (x) is selected from the group leucine, isoleucine, glycine and valine, and wherein (y) is selected from the group valine, leucine and serine; and altering the amino acid sequence comprising the sequence (x)D(y). In certain embodiments, the altering comprises at least one mutation of the amino acid sequence. In other embodiments, the amino acid sequence is removed. In particular aspects, the amino acid sequence comprises the sequence (x)D(y), wherein the (x)D(y) sequence is GDL, GDS, GDV, IDL, IDS, IDV, LDL, LDS, LDV, LDS, VDL or VDV. In certain more specific embodiments, the invention provides a modified proteinaceous composition that has altered, relative to the sequence of a native proteinaceous composition, at least one amino acid of a sequence comprising (x)D(y), wherein (x) is selected from the group leucine, isoleucine, glycine and valine, and wherein (y) is selected from the group valine, leucine and serine, for use as a medicament.
In certain aspects, the composition has a reduced ability to induce at least one toxic effect. In other aspects, the altering enhances the ability of the composition to induce at least one toxic effect. In particular embodiments, the toxic effect is, for example, VLS, the ability to induce apoptosis, a disintigrin-like activity, the ability to damage EC cells or a combination thereof. Of course; those of ordinary skill will, by following the teachings of this specification, be able to determine additional toxic effect that may be modulated according to the methods disclosed herein. In some embodiments of the invention, it is desirable to decrease the level of the toxic effect. For example, there is great benefit to be gained by creating an IT which exhibits no, or lessened, VLS. In alternative embodiments, it will be desirable to increase the level of a given toxic effect. For example, if a proteinacious compound is being used to induce apoptosis in a therapeutic manner, then modifying the compound to increase the level of apoptosis could be beneficial. Those of ordinary skill in the art will be able to determine any of a number of manners in which to employ the modulation of toxic effects that can be had with the methods of the invention.
In some embodiments, the (x)D(y) sequence comprises a residue on the surface of the composition. In certain aspects, the altering occurs at one or more (x)D(y) tri-amino acid sequences. In certain embodiments, the (x)D(y) sequence comprises at least one flanking sequence. In particular aspects, the altering the sequence comprises at least one alteration within the at least one flanking sequence. In certain aspects, the at least one flanking sequence is mutated. In some facets, the at least one flanking sequence is removed. In other facets, the alteration occurs within of from about 1 to about 6 residues of an (x) or an (y) of a (x)D(y) tripeptide sequence. In some aspects, the flanking sequence is C-terminal to the (x)D(y) sequence. In other aspects, the flanking sequence is N-terminal to the (x)D(y) sequence. In a particular facet, the at least one flanking sequence comprises two flanking sequences, wherein the two flanking sequences are N-terminal and C-terminal to the (x)D(y) sequence.
The proteinacious composition can be any presently known of discovered in the future that has the (x)D(y) tripeptide sequence. In some embodiments, the proteinaceous composition comprises a toxin, a cytokine, a viral sequence or a combination thereof. In particular aspects, the toxin comprises a plant toxin, a fungal toxin, a bacterial toxin, a RIP or a combination thereof. In certain facets, the toxin comprises Abrin A chain, Diphtheria Toxin (DT) A-Chain, Pseudomonas exotoxin, RTA, Shiga Toxin A chain, Gelonin, Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, Barley toxin or a combination thereof In other embodiments, the proteinaceous composition comprises a cytokine, such as, for example, Interleukin-2. In further embodiments, the proteinaceous composition comprises a viral sequence, such as, for example, an adenoviral sequence. In certain aspects, the proteinaceous composition further comprises, or is comprised in, an IT.
The invention, in some particularly preferred aspects, the invention provides a method of reducing the ability of a proteinaceous composition to promote VLS, comprising the steps of: identifying at least one amino acid sequence comprising the sequence (x)D(y), as defined above.
In certain aspects other aspects, the invention provides the use of a modified proteinaceous composition that has altered, relative to the sequence of a native proteinaceous composition, at least one amino acid of a sequence comprising (x)D(y), for the manufacture of a medicament for the treatment of a disease, including but not limited to GVHD, non-Hodgkin""s and Hodgkin""s lymphoma, myloma, as well as metastatic lesions of solid tumors and damage to endothelial cells (i.e., VLS).
The invention additionally provides a method of preparing an IT with a reduced ability to induce a toxic effect, comprising the steps of: identifying at least one amino acid sequence comprising the sequence (x)D(y); removing the amino acid sequence from the toxin; and conjugating the toxin to a composition comprising at least one antibody to produce an IT, wherein the IT produced possesses a reduced ability to promote a toxic effect when compared to a like IT wherein the amino acid sequence was not removed from the toxin.
The invention also provides a method of enhancing the ability of a proteinaceous composition to induce extravasation, comprising adding at least one amino acid sequence comprising (x)D(y) to the composition. In particular aspects, the composition comprises a peptide. In further aspects, the extravasation of the composition or at least one molecule is enhanced. In some embodiments, the composition is covalently conjugated to the at least one molecule. In certain facets, the extravasation of the molecule is enhanced. In additional facets, the molecule is a therapeutic agent, such as, for example, at least one IT, antibody, cytokine, virus or a combination thereof.
The invention further provides a method of reducing the toxic effects of a proteinaceous material in a patient, comprising administering to a patient a composition that mimics a sequence comprising (x)D(y). In particular aspects, the proteinaceous material comprises at least one (x)D(y) sequence. In additional aspects, the composition comprises at least one amino acid sequence comprising the sequence (x)D(y), wherein the amino acid sequence has been altered to possess an reduced ability to promote a toxic effect. In certain facets, the composition comprises at least one peptide.
The invention also provides a modified proteinaceous composition that has altered, relative to the sequence of a native proteinaceous composition, at least one amino acid of a sequence comprising (x)D(y), prepared according to the methods described above and elsewhere in this specification. In certain embodiments, the proteinaceous composition comprises a toxin, a cytokine, a viral sequence or a combination thereof. In certain aspects, the toxin is, for example, a plant toxin, a fungal toxin, a bacterial toxin, a RIP or a combination thereof. In additional aspects, the toxin comprises Abrin A chain, Diphtheria Toxin (DT) A-Chain, Pseudomonas exotoxin, RTA, Shiga Toxin A chain, Gelonin, Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, Barley toxin or a combination thereof. In other embodiments, the proteinaceous composition comprises a cytokine, such as for example, Interleukin-2. In other aspects, the proteinaceous composition comprises a viral sequence, such as, for example, an adenoviral sequence.
In certain facets, the composition further comprises an antibody. In particular aspects, the composition further comprises an IT. In additional facets, the IT further comprises at least a second agent, such as, for example, at least one effector molecule. In particular aspects, the effector molecule is a toxin, an anti-tumor agent, a therapeutic enzyme, an antiviral agent, a virus, a cytokine, a growth factor, or a combination thereof. In other facets, the agent is at least one reporter molecule.
The invention additionally provides an IT, comprising at least one proteinaceous molecule with a reduced ability to induce VLS, apoptosis, disintegrin-like activity or EC damage, wherein the proteinaceous molecule has at least one (x)D(y) or flanking sequence altered.
The invention provides a modified proteinaceous composition with an enhanced ability to promote extravasation, wherein the composition comprises at least one amino acid sequence comprising a (x)D(y) tripeptide or a flanking sequence relative to the native sequence.
In certain embodiments, the composition comprises a therapeutic agent, such as, for example, at least one IT, antibody, cytokine, virus or a combination thereof. In specific embodiments, the composition and the therapeutic agent are covalently conjugated. In particular facets, the composition is a therapeutic agent.
The invention also provides a RTA with a reduced ability to promote toxicity in a patient, wherein the (x)D(y) sequence comprising positions 74 to 76 is altered. In certain embodiments, the leucine at position 74 is altered, the aspartate at position 75 is altered, and/or the valine at position 76 is altered. In specific facets, the (x)D(y) sequence further comprises positions of from about 1 to about 6 residues of an (x) or an (y) of the (x)D(y) tripeptide sequence.
The invention also provides a method of reducing the ability of a proteinaceous composition to induce VLS, comprising the steps of: identifying at least one amino acid sequence (x)D(y); and altering, removing or mutating the amino acid sequence.
Flanking regions to the (x)D(y) tri-peptide sequence may be altered to reduce a proteinaceous composition""s ability to induce VLS. As used herein, a xe2x80x9cproteinaceous compositionxe2x80x9d refers to a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene, a polypeptide of greater than about 100 amino acids, and/or a peptide of from about 3 to about 100 amino acids, including peptides of 3, 4, 5, 6, etc., 10, 11, 12, 13, 14, etc., 20, 21, 22, etc, 30, 40, 50, 60, etc. 100, 110, 120, etc. 200, 220, 240, etc, 300, 350, 400, etc, 500, 600, 700, etc., and 1000 amino acids in length. In an aspect the C-terminal flanking amino acid sequence may be altered, mutated or removed if it is a threonine (T). In certain aspects, this method of altering this sequence and/or flanking amino acids is by removal of the amino acid sequence.
The invention provides a proteinaceous composition that has a reduced ability to induce VLS. In an aspect aspect, the proteinaceous composition that has been altered to remove at least one amino acid sequence contiguous with the composition comprising the sequence (x)D(y). In certain aspects of the present invention, the proteinaceous composition may be a ribosome-inactivating protein (RIP), including but not limited to gelonin, momordin, pokeweed antiviral protein (PAP), saporin, or trichosanthin; a toxin or toxin subunit, including but not limited to abrin A chain, diphtheria toxin (DT) A-chain, Pseudomonas exotoxin-A (PE38-lys), RTA, Shiga toxin A chain, or barley toxin; a cytokine including but not limited to IL-2. Proteins, polypeptides and/or peptides may be derived from RIPs, toxins or cytokines to be used in the methods and compositions of the present invention. In certain aspects, the proteinaceous composition may be used to make an IT with a reduced ability to promote or enhance VLS. In other aspects, the proteinaceous composition for use in an IT is a RIP and/or toxin sequence.
In other aspects of the present invention, proteins, peptides and/or polypeptides may be made that include the VLS-inducing sequence. These VLS-inducing proteinaceous compositions may be used to promote VLS, and increase the extravasation of molecules into tissues. In additional aspects of the present invention, proteinaceous compositions may be made that lack the VLS-inducing sequence. Such compositions may be used as inhibitors of agents that induce VLS in vivo or adenoviral vectors for gene therapy. The compositions of the present invention may be made by synthetic peptide synthesis or through the use of recombinant genetic technology, as would be known to those of ordinary skill in the art in light of the present disclosure. Toxin mutants lacking VLS activity can also be effective for protecting individuals against the native toxin.
As used herein the specification, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more. As used herein in the claim(s), when used in conjunction with the word xe2x80x9ccomprisingxe2x80x9d, the words xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more than one.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.